title: New methods for ALK status diagnosis in non-small-cell lung cancer: an improved ALK immunohistochemical assay and a new, Brightfield, dual ALK IHC-in situ hybridization assay.
abstract: BACKGROUND
  The demonstration of anaplastic lymphoma kinase (ALK) positivity in non-small-cell lung cancer (NSCLC) has been hindered by the technical complexity and interpretative challenges of fluorescence in situ hybridization methods for detection of ALK gene rearrangement and by the inadequate sensitivity of existing immunohistochemistry (IHC) methods for ALK protein detection. In this study, we sought to increase the sensitivity of ALK IHC detection and to develop a brightfield assay for concurrent detection of ALK protein expression and ALK gene rearrangement.
  
  
  METHODS
  We developed a horseradish peroxidase-based IHC detection system using the novel, nonendogenous hapten 3-hydroxy-2-quinoxaline (HQ) and tyramide. We also developed a dual gene protein ALK assay combining a brightfield break-apart in situ hybridization ALK assay with another sensitive IHC method using the novel, nonendogenous hapten 5-nitro-3-pyrazole. We examined the sensitivity and accuracy of these methods using surgically resected NSCLC cases examined with ALK fluorescence in situ hybridization.
  
  
  RESULTS
  The new HQ-tyramide IHC detection system offered readily interpretable staining with substantially greater sensitivity than conventional ALK IHC, and produced heterogeneous and homogeneous patterns of ALK protein staining among ALK-positive NSCLC surgical cases. The new 5-nitro-3-pyrazole-based IHC detection system was similar in ALK detection sensitivity to the HQ-tyramide IHC system and was compatible with the brightfield in situ hybridization assay.
  
  
  CONCLUSIONS
  The new HQ-tyramide IHC reagent system allows more sensitive assessment of ALK protein status in NSCLC cases. The new ALK gene-protein assay allows the concurrent visualization of ALK gene and ALK protein status in single cells, allowing more accurate ALK status determination even in heterogeneous specimens.
authors.affiliation: Medical and Scientific Affairs, Ventana Medical Systems, Inc., Tucson, Tucson, AZ 85755, USA. hiro.nitta@ventana.roche.com
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nitta
authors.forename: Koji
authors.initials: K
authors.lastname: Tsuta
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yoshida
authors.forename: Steffan N
authors.initials: SN
authors.lastname: Ho
authors.forename: Brian D
authors.initials: BD
authors.lastname: Kelly
authors.forename: Lauren B
authors.initials: LB
authors.lastname: Murata
authors.forename: Jerry
authors.initials: J
authors.lastname: Kosmeder
authors.forename: Katie
authors.initials: K
authors.lastname: White
authors.forename: Sandra
authors.initials: S
authors.lastname: Ehser
authors.forename: Penny
authors.initials: P
authors.lastname: Towne
authors.forename: Crystal
authors.initials: C
authors.lastname: Schemp
authors.forename: Abigail
authors.initials: A
authors.lastname: McElhinny
authors.forename: Jim
authors.initials: J
authors.lastname: Ranger-Moore
authors.forename: Chris
authors.initials: C
authors.lastname: Bieniarz
authors.forename: Shalini
authors.initials: S
authors.lastname: Singh
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Tsuda
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Grogan
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D002289:Carcinoma, Non-Small-Cell Lung / Q000737:chemistry*; D045744:Cell Line, Tumor; D006241:Haptens / Q000276:immunology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008175:Lung Neoplasms / Q000737:chemistry*; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis*
chemical_list: D006241:Haptens; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases